Methods and compositions for improving microvascular function, suppressing cyclooxygenase activity, reducing platelet aggregation and increasing levels of resveratrol in plasma

ABSTRACT

Methods of increasing the plasma concentration of resveratrol in a mammal in need thereof by administering to a mammal in need thereof an effective amount of trans-resveratrol and arginine for a period of at least two weeks.

BACKGROUND OF THE INVENTION Field of the Disclosure

The present disclosure generally relates to methods and compositionsthat involving resveratrol, which has been studied for its ability toreduce cardiovascular diseases. The present disclosure is on methods andcompositions involving trans-resveratrol and arginine, which provides avariety of benefits and advantages, including but not limited to a morebio-available and bio-active form of trans-resveratrol. The presentdisclosure also includes data from a study that demonstrates that, whencompared to other forms of trans-resveratrol, trans-resveratrol andarginine, provides various benefits and advantages, including but notlimited to, improving the absorption of resveratrol, increasing theamount of resveratrol in plasma levels, improving micro-vascularfunction, reducing platelet aggregation, suppressing cyclooxygenaseactivity, reducing platelet aggregation and increasing levels ofresveratrol in plasma.

General Background

Cardiovascular disease (hereafter, “CVD”) is a leading cause ofmortality in the United States and other industrialized nations [1, 2].Therefore, simple and effective methods to reduce risk of CVD areimportant for both personal and public health strategies. Evidence fromepidemiological investigations suggests that consumption of plant-basedfoods and products rich in polyphenolic compounds can havecardioprotective effect [3-5]. Among the polyphenols, a number ofcompounds have been suggested to have cardioprotective effects,including flavano-3-ols from cocoa [6], epigallocatechin gallate(hereafter, “EGCG”) from green tea [7], and resveratrol, a phenoliccompound found in grapes, red wine, purple grape juice, and some berries[8].

Resveratrol (3, 5, 4′-trihydroxystilbene) is a polyphenol, a stilbenoidaderivating from stilbene and is produced in plants. Resveratrol wasfirst isolated in 1940 from the roots of Veratrum grandiflorum, and thenin 1963 resveratrol from Polygonum cupsidatum, a plant that is used intraditional Chinese and Japanese medicine [9, 10]. However, resveratrolwas overlooked by the Western cultures until 1992 when it was suggestedto be an explanation for the “French Paradox”, an observation of lowerrates of CVD in the French population, which was attributed to higherred wine consumption [9]. The optimal health benefit derived from redwine is achieved at a consumption of more than five glasses a day forlonger period, which would be considerably more expensive than aresveratrol pill.

Resveraltrol was found well tolerated both in humans and animals.Resveratrol is non-genotoxic, non-mutagen, and has no reproductivetoxicity. Usual human dose is 0.8-33 mg/kg, bw/day, while animal studiesrevealed no toxicity effect even at the dose of 500 mg/kg, bw/day for 3months dosing [11-13]. Two structural isomers of resveratrol can befound in foods, cis- and trans- [8, 9]. The trans- form can undergoisomerisation to the cis- form when exposed to ultraviolet irradiation[14]. Trans-resveratrol (hereafter, “trans-res”) is biologically activeand the predominant form found in nature, and it is the isomer has beenused in supplements and clinical interventions [15], due to instabilityof the cis isomer [8]. (FIG. 1 shows the composition of trans-rescapsule). The naturally occurring amount of resveratrol in most foods isvery low. Thus, even though it can cause an effect in a relatively shortperiod of time, obtaining biologically active amount of resveratrol viadiet alone is difficult. Illustrative of this, typical daily intakes ofresveratrol from dietary sources are in the range of six to eight mg[16], while the amount of resveratrol supplementation in humans that hasbeen reported to result in increased brachial artery flow mediateddilatation (hereafter, “FMD”; a measure of vascular function) on anacute basis is 30 to 270 mg [17].

Although resveratrol demonstrated to have beneficial effects underexperimental conditions, most of its effects in humans are limited byits fast metabolism and low plasma exposure. Resveratrol undergoes rapidmetabolism in intestines and liver resulting in poor plasma exposure[18, 19]. Resveratrol is rapidly absorbed, and depending on the dose,reaches maximum peak plasma concentration anywhere from 30 min to twohours after intake [15, 20]. Absorbed resveratrol is rapidly metabolizedto conjugates of sulfate and glucuronide, and the metabolites appear tobe quickly cleared from circulation [8, 15, 21].

Despite its rapid absorption and metabolism, supplemental resveratrolcan have positive effects on a number of cardiovascular health outcomes,including endothelial function, platelet function, and blood lipids [15,22]. An enhanced FMD response was reported with resveratrolsupplementation as low as 10 mg per day for three months [23], as wellas 75 mg for six weeks [24]. Reduction in low density lipoprotein(hereafter, “LDL”) cholesterol was shown after daily intake of 8 mg ofresveratrol for six months [25], while a decrease in total cholesterolwas observed after supplementation of 250 mg for three months [26].Daily supplementation with 8 mg of resveratrol for six months, followedby 16 mg per day for additional six months, resulted in a significantdecrease in plasminogen activator inhibitor type 1 (hereafter, “PAI-1”),which is known to promote hemostasis [27], and is secreted by activatedendothelial cells [28].

Resveratrol has been extensively studied for its potential to improvehealth and longevity. Prospective benefits of resveratrolsupplementation have led to annual sales of more than $30 million in theUnited States in 2010. However, resveratrol is very expensive and limitsthe use of high dose. The inventions of the present disclosure providean improved form of resveratrol which provides benefits including butnot limited to improved bioavailability and pricing [29, 30].

A novel resveratrol-arginine conjugate (hereafter, “ResArgin™”; wasprovided by Gateway Health Alliances, Inc. Fairfield, Calif., hereafter,“Gateway”. ResArgin™ composition is shown in FIG. 2. As part of thepresent disclosure, ResArgin™ was studied in rats, demonstrating, amongother things, higher peak plasma levels and total plasma levels for alonger period of time when compared to trans-res and two otherresveratrol conjugates: resveratrol-citrulline and resveratrol-succinate[31]. (See FIG. 3)

Additional experiments were done to assess, among other things, thebioavailability and bioactivity of ResArgin™ among postmenopausal women,a population at risk for cardiovascular disease [32]. The studydetermined, among other things, whether ResArgin™ intake would improvevascular function and compared those results to those of trans-res. Thestudy also determined whether ResArgin™ improves measures of plateletfunction more than trans-res. Lastly, the study analyzed whether plasmaresveratrol levels would be greater with ResArgin™ supplementation whencompared to trans-res and that the level of improvement would beunexpected and related to the magnitude of change in the markers ofvascular function and platelet function.

SUMMARY

BRIEF DESCRIPTION OF DRAWINGS

The drawings disclose illustrative embodiments and represent graphicalsummaries of the data explained and described herein. They do not setforth all embodiments. Other embodiments may be used in addition orinstead. Details that may be apparent or unnecessary may be omitted tosave space or for more effective illustration. Conversely, someembodiments may be practiced without all of the details that aredisclosed. When the same numeral appears in different drawings, it isintended to refer to the same or like components or steps.

The above-mentioned features and objects of the present disclosure willbecome more apparent with reference to the following description takenin conjunction with the accompanying drawings and in which:

FIG. 1 lists the composition of trans-res capsule used in theexperiments and studies discussed herein.

FIG. 2 lists the composition of ResArgin™ in the capsule used in theexperiments and studies discussed herein.

FIG. 3 is a table which graphically compares ResArgin™ with otherresveratrols in other pharmacokinetic rat studies. The resultdemonstrates that ResArgin™ had higher levels of plasma concentrationwhen compared to other indicated resveratrols.

FIG. 4 is a table which graphically compares ResArgin™ with otherresveratrols in other pharmacokinetic rat studies. The resultdemonstrates that ResArgin™ resulted in higher plasma concentration ofresveratrol when compared to other indicated resveratrols.

FIG. 5 is a line chart which demonstrates that ResArgin™ had higherplasma concentration and persisted for a longer period when compared totrans-resveratrol alone, a physical mixture of resveratrol and arginine,and two other resveratrol conjugates: resveratrol-citrulline andresveratrol-succinate.

FIG. 6 is a flow chart that describes the process of the study discussedherein. 1 hr, one hour; 2 h, two hour.

FIG. 7 is a flow chart that describes the number of the participants andprocess in each separate study discussed herein.

FIG. 8 is a table which describes the baseline characteristics of studyparticipants. Values are presented as mean±standard error of the mean(hereafter, “SEM”); 1 hr, one hour study; 2 hr, two hour study; BMI,body mass index; SBP, systolic blood pressure; DBP, diastolic bloodpressure; MAP, mean arterial pressure; Glc, glucose; Chol, cholesterol;HDL, high density lipoprotein; LDL, low-density lipoprotein; TG,triglyceride.

FIG. 9 is a bar chart which graphically describes the changes(Δ=post−pre) in microvascular function (reactive hyperemia index,hereafter, “RHI”; also termed peripheral arterial tonometry index) forone hour study. The results demonstrates that change in RHI wassignificantly greater with ResArgin™ for one hour study (0.123±0.121trans-res versus 0.527±0.158 ResArgin™, p=0.035). *p<0.05 differencebetween treatments. Statistical analysis was performed using pairedt-test.

FIG. 10 is a bar chart which graphically describes the changes(Δ=post−pre) in microvascular function (Framingham Reactive HyperemiaIndex, hereafter, “FRHI”) for one hour study. The results demonstratesthat change in FRHI was significantly greater with ResArgin™ for onehour study (0.154±0.062 trans-res versus 0.396±0.070 ResArgin™,p=0.009). *p<0.05 difference between treatments. Statistical analysiswas performed using paired t-test.

FIG. 11 is a bar chart which graphically describes the changes(Δ=post−pre) in microvascular function (RHI) for two hour study. Theresults demonstrates that change in RHI was not significantly betweentreatments for two hour study (−0.108±0.161 trans-res versus 0.306±0.173ResArgin™; p>0.05).

FIG. 12 is a bar chart which graphically describes the changes(Δ=post−pre) in microvascular function (FRHI) for two hour study. Theresults demonstrates that change in FRHI was not significantly differentbetween treatments for two hour study (0.113±0.042 trans-res versus0.216±0.081 ResArgin™; p>0.05).

FIG. 13 is a bar chart which combines the data from one and two hourstudies in changes of RHI. The result demonstrates that a significantimprovement in RHI was seen one hour, but not two hours, after intake ofResArgin™ (2.119±0.085 at baseline versus 2.715±0.116 at 1 hour(p<0.0001) and 2.383±0.124 at 2 hours). a,b Values with differentsuperscript letters are significantly different, p<0.05. Statisticalanalysis was performed using Univariate ANOVA with Bonferroni post hoc.

FIG. 14 is a bar chart which combines the data from one and two hourstudies in changes of FRHI. The result demonstrates that a significantimprovement in FRHI was seen one and two hours after intake of ResArgin™(0.667±0.044 at baseline versus 1.095±0.061 at one hour (p<0.0001) and0.882±0.065 at two hours (p=0.020). Additionally, the difference betweenone hour and two hours with ResArgin™ did not quite reach significance(p=0.052). No differences were observed with trans-res. a,b Values withdifferent superscript letters are significantly different, p<0.05.Statistical analysis was performed using Univariate ANOVA withBonferroni post hoc.

FIG. 15 is a bar chart which describes the changes in ResArgin™ plasmaresveratrol contentration in one hour study. The result demonstratesthat resveratrol plasma concentration significantly increased frombaseline at 30 and 60 minutes after intake of ResArgin™ (0.00 (0.00,0.00) at baseline versus 0.83 (0.21, 2.89) at 30 minutes versus 2.09(1.60, 3.63) at 60 minutes; p<0.001). *p<0.05 compared to baseline.Statistical analysis was performed using non-parametric Friedman's 2-wayANOVA by ranks test (n=30). Values are presented as median(interquartile range).

FIG. 16 is a bar chart which describes the changes in trans-res plasmaresveratrol contentration in one hour study. The result demonstratesthat resveratrol plasma concentration significantly increased frombaseline at 30 and 60 minutes after intake of trans-res 0.00 (0.00,0.00) at baseline versus 2.04 (0.82, 2.54) at 30 minutes versus 2.65(1.69, 3.44) at 60 minutes; p<0.001). *p<0.05 compared to baseline.Statistical analysis was performed using non-parametric Friedman's 2-wayANOVA by ranks test (n=30). Values are presented as median(interquartile range).

FIG. 17 is a line chart which graphically describes the changes inplasma resveratrol concentration for both ResArgin™ and trans-res. Theresult demonstrates that both treatments were not significantlydifferent.

FIG. 18 describes the changes in platelet function for intake of bothResArgin™ and trans-res. The results revealed a significant increase inlag time one-hour post consumption with both treatments when plateletswere stimulated with 1 μL collagen. Also, a significant decrease inslope was seen one hour after consumption of ResArgin™, but not for thetrans-res group. Stimulation with 5 μL collagen showed no significantchanges in either group. Significant decreases in maximum aggregationand area under the curve were observed one hour after intake ofResArgin™ when platelets were stimulated with 10 μL arachidonic acid,but no changes were noted in the trans-res group. Lumi-aggregometry with10 μL ADP revealed a significant decrease in slope one-hour postconsumption of both trans-res and ResArgin™. Values are presented asmedian (interquartile range). ADP=adenosine diphosphate; AA=Arachidonicacid; Max aggregation=maximum aggregation; AUC=area under the curve;ns=not significant. p<0.05 represent statistically significantdifference between the baseline measurement and the 1-hourpost-consumption of the assigned treatment (n=31). Statistical analysiswas performed using non-parametric Friedman's 2-way ANOVA by ranks test.

FIG. 19 is a table which compares resveratrol conjugates with twoSirtris formulations. Sirtris is a GSK Company, data is from patent WO2009/089011 A2; units of the parameters normalized for comparativepurpose. The results demonstrate that ResArgin™ from Gateway had higherCmax (11289 nM) than Sirtris formulations (7416 nM and 4671 nM).

FIG. 20 is a bar chart which compares ResArgin™ among other resveratrolin terms of aqueous solubility. The result demonstrates that ResArgin™improved its solubility.

FIG. 21 is a bar chart which compares ResArgin™ among other resveratrolin terms of aqueous solubility. The result demonstrates that ResArgin™improved its solubility.

METHODS

The study was a randomized, controlled, double-blind, crossover trialthat postmenopausal women were randomly assigned to consume 90 mg ofResArgin™ or trans-res , in a crossover design, at least one week apart,after an overnight fast. The study included two separate investigations:a one hour study and a two hour study (FIG. 1). Based on the results,dosage of 10 mg to 5000 mg ResArgin or trans-res daily for a period ofat least two weeks are expected to have identical results. The one hourstudy assessed changes in select outcomes 30 minutes and one hourpost-consumption of the resveratrol treatments. The two hour study wasdesigned to fully assess and compare the responses to trans-res andResArgin™.

Results

A more rapid and pronounced response from ResArgin™ than trans-res onmarkers of microvascular function. The changes noted were significantlyassociated with plasma resveratrols levels. At one hour, significantincreases RHI and FRHI. Significant reductions in platelet reactivityunder certain test conditions were noted at one hour after ResArgin™intake, but not for trans-res.

Plasma resveratrol levels were significantly increased 30 and 60 minafter the consumption of both ResArgin™ and trans-res, but the levelswere only correlated with RHI and FRHI in the ResArgin™ group.

DETAILED DESCRIPTION

Participants of Study

Healthy postmenopausal women ages 50 to 70 years of age were recruitedfrom the greater Sacramento area via email and newspaper advertisements.Postmenopausal status was defined as the cessation of the menses for atleast one year, and a level of follicular stimulating hormone(hereafter, “FSH”) of 23-116.3 mIU/mL. All women were non-smokers, hadno history of chronic disease, had no allergies to fruits, and did notregularly use any medications except thyroid medications. Use of dietarysupplements other than standard multivitamin/mineral formulas (supplyingup to 100% daily value) was an exclusion factor. Eligibility wasdetermined by completion of a telephone interview, followed by aclinical screening visit conducted at the UC Davis Ragle Human NutritionResearch Center, which included a peripheral arterial tonometry(hereafter, “PAT”) measurement, comprehensive metabolic and lipidpanels, and assessment of platelet function abnormalities as determinedby platelet function analyzer-100 (PFA-100) closure time readings(94-193 sec).

For the one hour study, 37 women were enrolled between March and October2012, of which 34 finished (FIG. 7). The two hour study was conductedbetween June and November 2014, of which 27 women were enrolled andcompleted the study (FIG. 7).

The Institutional Review Board of the University of California, Davisapproved the study protocol, and all participants provided writteninformed consent prior to enrollment.

Study Design

Participants were randomized by block design to consumed trans-res orResArgin™ in random order. Randomization was performed by the studycoordinator following a predetermined plan formulated via a web-basedrandom number generator (www.randomization.com). Both treatmentscontained 90 mg of trans-res, provided as two solid white, hard-shelledcapsules (FIG. 1-5). The study included two separate investigations: aone hour study and a two hour study (FIG. 6). Baseline anthropometricand biochemical measures collected during test day one are listed inFIG. 8. During each study visit, which took place after an overnightfast, a baseline measurement of PAT and a blood sample were collected,followed by consumption of the assigned treatment. During the one hourstudy, PAT measurements and blood samples were collected thirty andsixty minutes after the capsules were consumed. During the two hourstudy, a PAT measurement and blood sample were taken two hours after thecapsules were consumed.

All participants completed a three-day food record, which included twoweekdays and a weekend day, one week prior to the first test day. Forthe duration of the study period, the participants were instructed tocontinue their normal dietary patterns while avoiding the intake ofresveratrol-rich foods such as red wine, red grapes, peanuts andberries. Food records were analyzed using the Food Processor SQLsoftware (version 10.1.0).

Data Analysis

All data are expressed as mean SEM, unless otherwise stated. Data wereinitially assessed for normality and outliers, and values not normallydistributed, determined by the Shapiro-Wilk test, were transformed andrechecked for normality. Statistical analyses were conducted usingunivariate ANOVA with a Bonferroni post-hoc confidence interval or apaired t-test. Two variables were used: Treatment (trans-res andResArgin™) and Time (0, 1 and/or 2 hours). For nonparametric data,changes from baseline and differences between the treatments wereanalyzed using Friedman's two-way analysis of variance by ranks with theWilcoxon signed-rank post-hoc test and presented as medians(interquartile range). P-values of <0.05 were considered statisticallysignificant. All analyses were performed with IBM SPSS software (version22.0.0.0).

Metabolic Measurements

Blood samples were analyzed for a comprehensive metabolic panel, lipidpanel, and complete blood count by the UC Davis Medical CenterDepartment of Pathology.

Vascular Function

Microvascular function was assessed via PAT, using the Endo-PAT2000(Itamar Medical Ltd., Caesarea, Israel) [33]. Briefly, prior to the PATmeasurement, participants were acclimated to the controlled test roomconditions by resting in a supine position for 30 minutes. A fingerprobe was then placed on the middle finger of both hands, and a bloodpressure cuff was fitted on the forearm of the experimental arm (thenon-dominant arm). The measurement was performed in a supine positionwith both arms supported at heart level. The procedure included five toten minutes of baseline recording, followed by a five-minute occlusionperiod during which the blood pressure cuff was inflated approximately60 mmHg above the individual's systolic blood pressure. After fiveminutes, the pressure was released and the resulting reactive hyperemiaresponse recorded for an additional three to five minutes. The systemsoftware then automatically calculated three indices of microvascularfunction.

First, the reactive RHI was calculated, which is the ratio of theaverage of the pulse wave amplitude (hereafter, “PWA”) during aone-minute period following one minute of reactive hyperemia to theaverage PWA during a 3.5-minute of baseline period. An increase in RHIindicates improvement of microvascular function. Second, the FRHI wascalculated as the natural logarithmic transformation of the RHI ratio,without the baseline correction factor and utilizing only the readingsfrom 90 to 120 seconds following the reactive hyperemia. Third, anaugmentation index (hereafter, “AI”) was calculated as a measure ofarterial stiffness, which was considered along as well as normalized toa heart rate of 75 beats per minute (hereafter, “AI@75”). The AI andAI@75 are considered as supplementary measures of cardiovascular riskassessment, since an increase in AI represents an increase incardiovascular risk [34].

One Hour Study

Changes (Δ=post−pre) in microvascular function for RHI and FRHI weresignificantly greater with ResArgin™ compared to trans-res (FIGS. 9,10). For RHI, change with ResArgin™ for 1 hour study (0.527±0.158,p=0.035) was significantly greater with trans-res (0.123±0.121,p=0.035). As for FRHI, change in ResArgin™ was also significantlygreater than it in trans-res (0.306±0.173 versus −0.108±0.161; p>0.05).

Two Hour Study

Changes (Δ=post−pre) in microvascular function were not significantlydifferent between both treatments in RHI (−0.108±0.161 trans-res versus0.306±0.173 ResArgin™; p>0.05). Similarly, changes in microvascularfunction were not significantly different between both treatments inFRHI (0.113±0.042 trans-res versus 0.216±0.081 ResArgin™; p>0.05) in thetwo hour study (FIGS. 11, 12).

Combined Data

Univariate analysis revealed a significant interaction between treatmentand time (p=0.020) for RHI. Further analysis showed a significantimprovement in RHI one hour after intake of ResArgin™ (2.715±0.116,p<0.0001) compared to baseline (2.119±0.085) (FIG. 13), which was notobserved with trans-res. Similarly, a significant treatment and timeinteraction was seen for FRHI at one hour for the ResArgin™ groupcompared to their baseline values (0.667±0.044), while no significantdifferences were noted in the trans-res group. Significant improvementsin FRHI were seen one and two hours post-consumption of ResArgin™(1.095±0.061, p<0.0001 and 0.882±0.065 p=0.020, respectively; FIG. 14),while no changes were noted for the trans-res group. The differencebetween one hour and two hour FRHI values with ResArgin™ showed a strongtrend (p=0.052), while the difference did not quite reach significance.No significant changes were noted for AI or AI@75 in either group.

Plasma Resveratrol Concentrations

To assess bioavailability, plasma resveratrol levels were analyzed witha newly developed analytical method, because existing methods formeasurement of resveratrol are focused on wine and other botanicalsources and do not consider factors in plasma such as albumin. Plasmaresveratrol concentrations were determined following analytical methodsused to assess flavan-3-ols in human plasma following intake of astandardized cocoa extract [35]. Briefly, plasma was treated withacidified methanol [0.5% (v/v) of acetic acid in methanol; precooled to−20° C.] containing an appropriate recovery standard. The mixture wasstored at −80 C for 12 hours to allow for cryo-assisted proteinprecipitation, and then centrifuged for 15 min at 16,500×g. Thesupernatant fraction was transferred and its volume reduced to less than50 μL by removing the solvents under vacuum with a 7 SpeedVacConcentrator (Thermo Electron Corporation, Milford, Mass.). Fifty μL ofacidified methanol was then added to the concentrated sample extracts,which were flushed with argon and stored at −80° C. until analysis usingan Agilent high-pressure liquid chromatography (hereafter, “HPLC”) 1100series unit (Agilent Technologies, Santa Clara, Calif., USA). Afterthawing, 150 ul of HPLC grade water containing an appropriate internalstandard was added to the samples.

The plasma resveratrol concentrations were not significantly differentbetween ResArgin™ and trans-res. After intake of ResArgin™, plasmaresveratrol concentration significantly changed from baseline ((0.00(0.00, 0.00) to 30 minutes (0.83 (0.21, 2.89)) and 60 minutes (2.09(1.60, 3.63); p<0.001; FIG. 15), as well as after trans-res intakeintake (0.00 (0.00, 0.00) at baseline versus 2.04 (0.82, 2.54) at 30minutes versus 2.65 (1.69, 3.44) at 60 minutes; p<0.001; FIG. 16). Meansline graph for the two treatment are shown below in FIG. 17.

Correlations

A significant positive correlation was noted between plasma resveratroland RHI (rs=0.221; p=0.034) as well as FRHI (rs=0.348; p=0.001). Furtheranalysis showed a significant correlation between plasma resveratrol andRHI and FRHI for ResArgin™ (rs=0.435; p=0.002 and rs=0.527; p<0.0001,respectively) but not between plasma resveratrol and RHI and FRHI fortrans-res (rs=0.010; p=0.949 and rs=0.165; p=0.273, respectively).

Platelet Function

Platelet reactivity during clinical screening was assessed by a PFA-100(Siemens, Deerfield, Ill.) [36], which measures platelet function bysimulating arterial hemostasis under blood flow shear stress. During theintervention, platelet function was assessed by a whole blood/opticallumi-aggregometer (Chrono-log Model 700, Havertown, Pa.) [37, 38].Briefly, platelet aggregation was measured via electrical impedence (orelectrical resistance), whereas lumi-aggregometry was used to examineaggregation together with adenosine triphosphate (hereafter, “ATP”)release, which is determined with firefly luciferin-luciferase system.For the impedance technique without ATP release, 500 μL of each, salineand whole blood were added to a plastic cuvette and stirred at 1000 rpm.For the lumi-aggregometry procedure, 450 μL saline, 450 μL whole bloodand 100 μL of luciferin-luciferase reagent were added to a plasticcuvette and stirred at 1000 rpm. One of the following agonists was usedto stimulate platelets: collagen (1 μL and 5 μL), AA (10 μL) or ADP (10μL). ATP release was measured only when platelets were activated withADP. The aggregation was measured over a period of six minutes,following the addition of an agonist.

Platelet function analysis revealed a significant increase in lag timeone-hour post consumption with both treatments when platelets werestimulated with 1 μL collagen (p=0.008 for trans-res and p=0.004 forResArgin™; FIG. 18). A significant decrease in slope was seen one hourafter consumption of ResArgin™ (p=0.004; FIG. 18), but not for thetrans-res group. Stimulation with 5 μL collagen showed no significantchanges in either group. Lumi-aggregometry with 10 μL ADP revealed asignificant decrease in slope one-hour post consumption of bothtrans-res and ResArgin™ (p=0.008 and p=0.014, respectively; FIG. 18).Significant decreases in maximum aggregation and AUC were observed onehour after intake of ResArgin™ when platelets were stimulated with 10 μLAA (p=0.041 and p=0.005, respectively; FIG. 18), but no changes werenoted in the trans-res group.

Discussion

The present study assessed, among other things, the relative bioactivityand bioavailability of a supplemental ResArgin™ compared to trans-res,the form commonly used in most dietary supplements. The data supportedimprovements in microvascular function and platelet function withResArgin™ which were likely correlated with increases in plasmaresveratrol levels, while no effects were noted following intake oftrans-res.

Resveratrol is reported to support endothelial nitric oxide synthase(hereafter, “eNOS”) [39] and endothelial cell function. Endothelialdysfunction is an initial step in atherosclerosis [40], and ischaracterized by reduced nitric oxide (hereafter, “NO”) bioavailability,an important vasodilator that is directly produced and released byendothelial cells [41, 42], which is involved in initiation andprogression of atherosclerosis, a multifaceted condition resultingpathologies associated with CVD. Treatment of human umbilical veinendothelial cells with a physiological concentration of trans-res (1 μM)up-regulated the expression of eNOS [43], although cell culture modelsmay not accurately reflect in vivo expression due to the lack ofmetabolites in the culture medium. Consequently, examining ifsupplementation with resveratrol can affect microvascular function andindicators of NO activity is important.

A number of investigations have been explored in a potential value ofresveratrol supplementation on vascular function, since vasodilation ishighly dependent on NO bioavailability. The measurement is primarilyconducted by FMD, which assesses NO-mediated response in the conduitarteries of the peripheral circulation. However, evidence on this topicis conflicting. Supplementation with 600 mg of red grape extract led toa significant peak in FMD response 60 minutes post-consumption whencompared to baseline measurements and a placebo [44]. It should be notedthough, that in addition to 0.9 mg of trans-res the supplement evaluatedin the above study contained an array of grape polyphenols, some in muchhigher concentrations than resveratrol (4.32 mg epicatechin, 2.72 mgcatechin and 2.07 mg gallic acid), which may have contributed to thepositive effects on endothelial relaxation. A recent publication thatinvestigated the bioactivity of multiple supplements, including 100 mgof resveratrol, 800 mg each of green, black, and white tea extract, 250mg of pomegranate extract, 650 mg of quercetin, 500 mg ofacetyl-l-carnitine, 600 mg of lipoic acid, 900 mg of curcumin, 1 g ofsesamin, 1.7 g of cinnamon bark extract, and 1.0 g fish oil for sixmonths showed no significant change in FMD [45]. A study testing asupplement containing 100 mg of resveratrol combined with vitamin D3,quercetin and rice bran phytate and microencapsulated in plant starchesand dextrins (Longevinex; Resveratrol Partners, LLC) for three monthsreported improvements in FMD [39]. Because vitamin D, quercetin, andrice bran phytates have positive effects on cardiovascular outcomes ontheir own [39], it is difficult to determine if the positive changeswere due to resveratrol, synergism with other compounds, or the othercompounds independent of the resveratrol. An intervention that examinedthe effects of a single dose or 30, 90, and 270 mg of trans-res one hourafter consumption reported an increase in FMD with all three levels[17]. Wong et al. also reported that intake of 75 mg of trans-res led toa significant increase in FMD one hour post-consumption, as well asafter daily intake for six weeks [24]. Similarly, a study thatsupplemented stable coronary artery disease patients with 10 mg oftrans-res for three months demonstrated a significant improvement in FMD[23]. In contrast, a study that assessed microvascular function by PATreported no significant changes in RHI (PAT index) 90 minutes afterconsumption of a supplement containing 1, 1.5 or 2 g of trans-res (datawere combined from the three doses) that was served with a meal.However, a trend towards improvement (p=0.06) was seen in the RHI afterfour weeks of daily intake compared to the baseline visit [46]. A recentinvestigation reported no effect of daily intake of 250 mg of trans-resfor eight weeks, but a positive effect in the control group [47].Interestingly the same study reported that supplementation withtrans-res abolished the positive effects of daily exercise on bloodlipids [47].

The exemplary dose of resveratrol used in the present study (90 mg) waspreviously reported to induce positive effects on endothelialrelaxation, measured by FMD, at one hour after intake [17]. This studyhas witnessed an improvement in changes of microvascular function (RHI),one hour after consumption of ResArgin™ but not with trans-res. Combineddata from the one hour and two hour studies also revealed a significantimprovement in the RHI one hour after intake. The change in RHI was notsignificantly different between the treatments at the two-hour timepoint, which suggests an acute response within one hour after intake ofResArgin™ that appears to diminish by two hours after intake. Analysisof FRHI data confirms this argument, since the change in FRHI wassignificantly greater with ResArgin™ at the one-hour time point, and notat two hours. Findings from this study are also in agreement with thepreviously mentioned investigation [46] that reported no significantchanges in microvascular function measured by PAT acutely with unalteredtrans-res.

In addition to microvascular function, a number of investigations haveexamined resveratrol bioavailability using a wide range of dosages, andinconsistent findings have been suggested as the reason for conflictingphysiological responses [21]. Multiple factors are known to influenceresveratrol bioavailability, including the food matrix, dose andphysical properties of the molecule, all of which have the potential toalter maximal plasma concentration (hereafter, “Cmax”) and the half-life[15, 21]. Efforts have been made in the past years to increaseresveratrol bioavailability by enhancing the absorption rate and/orreducing intracellular metabolism to increase circulating levels ofresveratrol [21]. For example, a recent study reported that intake of a2 g lozenge containing 46% ribose, 46% of a fructose/sucrose mixture,and 8% trans-res led to a peak plasma concentration 15 minutes afterconsumption [48]. Evidence from this study showed that Cmax was achievedat an accelerated rate compared to previously reported averages at 30minutes to two hours [20] for trans-res supplements. Two majorlimitations of this study are the small sample size and the lack ofdetails regarding the plasma analysis methods. A study that supplementedhealthy volunteers with a form of trans-res, where 40 g of resveratrolwas solubilized in a lipid solution, led to a significantly higher Cmaxwhen compared to trans-res alone [49]. However, it is unknown if ahigher Cmax is related to clinical efficacy [21]. A recent report notedthat supplementation with various doses of trans-res (0.073 mg to 5 g)also led to quick absorption and elevations of plasma resveratrol levels[15], but independent of clinical outcomes, it is difficult to interpretthese results.

In another attempt to assess bioavailability, a significant increase inthe urinary excretion of resveratrol metabolites was reported fourhours, as well as 15 days, after intake of 187 mg of a beverage thatcontained 280 μg/L of hydroxycinnamic acids, 16 mg/L of anthocyanins, 96mg/L of flavanols, 83 mg/L of hydroxybenzoic acids, and 5.7 mg/L ofstilbenes, compared to baseline measurements or the control treatment[50]. Interestingly, this intervention also reported that sex might havean effect on bioavailability, as higher excretion of resveratrolmetabolites were noted in women than men [50]. A previous interventionwith postmenopausal women showed an increase in plasma resveratrolconcentration after supplementation of 75 mg of trans-res for twelveweeks, although, no significant changes in plasma lipids or inflammatorymarkers, including C-reactive protein (hereafter, “CRP”) andinterleukin-6 (hereafter, “IL-6”), were noted [29].

Plasma resveratrol concentrations in the study were not significantlydifferent between the two treatments at 30 and 60 minutes after intake.However, plasma resveratrol levels do not take into consideration thatresveratrol might be present in various cells (e.g., erythrocytes) ortissues, and thus not measured in plasma [15, 21]. Additionally, thedata showed an upward sloping curve for trans-res and ResArgin™ at 30and 60 minutes, and it is unknown where the Cmax would have beenachieved for each treatment. It is also unknown if Cmax or the areaunder the curve are the best indicators of bioavailability, and morestudies are needed to clarify this issue.

A significant positive association is seen between plasma resveratroland RHI, as well as FRHI. This finding is in agreement with the reportedimprovements in FMD related to an increase in plasma resveratrolconcentrations [17]. Further analysis demonstrated that plasmaresveratrol levels were not significantly correlated with RHI and FRHIfollowing trans-res intake, but were significantly correlated betweenplasma resveratrol and RHI and FRHI following intake of ResArgin™.

One possible explanation for the improved bioactivity of ResAgr comparedto trans-res would be a synergistic effect between resveratrol andarginine. It is unlikely that arginine alone was responsible for thepositive effects, since the ResArgin™ supplement provided 80 mg ofarginine, a relatively small amount compared to the reportedimprovements in vascular function with arginine supplementation,including two hours post-consumption of 15 g of L-arginine [51], andafter daily intake of 21 g for three days [52]. However, the amount ofarginine in ResArgin™ could have had an effect on the metabolism ofResArgin™, preserving resveratrol in a more bioactive form.

With respect to platelet function, since platelets are among the firstto arrive at the site of endothelial activation [53]. But the evidenceon the effects of resveratrol on platelet function is scarce. Moderateintake of red or white wine (300 mL/d) for 15 days resulted in anincrease of resveratrol plasma levels, and a significantly higherrelease of NO by stimulated platelets [54]. Daily trans-ressupplementation (10 mg) for three months significantly reduced plateletaggregation in stable coronary artery disease patients [23]. In vitrotreatment of platelets with trans-res (10-1,000 μM) significantlyreduced aggregation in a dose dependent fashion, while resveratrolsupplementation (4 mg/kg/day) inhibited ADP-induced platelet aggregationin vivo in a hypercholesterolemic rat model [55]. Additional evidencefrom in vitro studies suggests that trans-res can inhibit cyclooxygenase(hereafter, “COX”) activity and thus reduce platelet aggregation [56,57]. Cyclooxygenase is an enzyme that converts AA to thromboxane A2(hereafter, “TXA2”), and when this enzyme is inhibited, a significantreduction in platelet aggregation is noted [58]. Findings from our studyare in agreement with the evidence on the effects of resveratrol onplatelet function. The results showed that supplementation withResArgin™ resulted in a significant decrease in maximal aggregation andAUC when platelets were stimulated with AA. This suggests thatsupplementation with ResArgin™ may have suppressed COX activity.

The study has certain limitations. Due to equipment or operator failure,the sample size that was originally estimated was not fully achieved,although a sufficient number of measurements were collected to obtainstatistical significance. The study population of postmenopausal women,for whom age and postmenopausal status were risk factors for CVD, wasunique, and it would be of interest to examine if similar effects wouldbe seen in different at-risk populations such as overweight or obeseadults or children, among those with diabetes, or smokers. Since onlyfemales were studied, findings are limited in making generalizations tomen regarding bioavailability and bioactivity. Including multiple timepoints, both short-term and chronically, would better define thekinetics of the vascular response, platelet reactivity and plasma levelswe observed. Additionally, examination of plasma NO bioavailability mayprovide a better understanding of changes in endothelial function seenwith ResArgin™.

In conclusion, the data reports significant improvements inmicrovascular function when 90 mg of resveratrol was consumed asResArgin™ but not as trans-res. The changes noted may have beensignificantly associated with plasma resveratrol levels. Lastly, AAinduced platelet aggregation was reduced with ResArgin™ but not withtrans-res. Collectively, evidence from this intervention suggests thatsupplementation with ResArgin™ might have greater cardioprotectivebenefits than trans-res.

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1. A method of increasing the plasma concentration of resveratrol in amammal in need thereof, the method comprising: providing to a mammal inneed thereof 10 mg to 5000 mg daily of a trans-resveratrol and arginineconjugate for a period of at least two weeks. 2-3. (canceled)
 4. Themethod according to claim 1, wherein the effective amount of thetrans-resveratrol and arginine conjugate is 50 mg to 900 mg daily. 5.The method according to claim 1, wherein the effective amount of thetrans-resveratrol and arginine conjugate is 50 mg to 500 mg daily. 6.The method according to claim 1, wherein the period of at least twoweeks is at least four weeks.
 7. A method comprising: providing a mammalin need of increased plasma concentration of resveratrol 10 mg to 5000mg daily an cffcctivc amount of a trans-resveratrol and arginineconjugate for a period of at least two weeks. 8-9. (canceled)
 10. Themethod according to claim 7, wherein the effective amount of thetrans-resveratrol and arginine conjugate is 50 mg to 900 mg daily. 11.The method according to claim 7, wherein the effective amount of thetrans-resveratrol and arginine conjugate is 50 mg to 500 mg daily. 12.The method according to claim 7, wherein the period of at least twoweeks is at least four weeks.
 13. A method comprising: administering toa mammal in need of increased plasma concentration of resveratrol 10 mgto 5000 mg daily of a trans-resveratrol and arginine conjugate for aperiod of at least two weeks. 14-15. (canceled)
 16. The method accordingto claim 13, wherein the effective amount of the trans-resveratrol andarginine conjugate is 50 mg to 900 mg daily.
 17. The method according toclaim 13, wherein the effective amount of the trans-resveratrol andarginine conjugate is 50 mg to 500 mg daily.
 18. The method according toclaim 13, wherein the period of at least two weeks is at least fourweeks.
 19. The method according to claim 13, wherein the period of atleast two weeks is at least six weeks.